Reverse transcriptase (RT) is an enzyme required for replication of the Human Immunodeficiency Virus (HIV) and is the target of most of the therapeutic agents approved for clinical use in the treatment of AIDS. The majority of the drugs that have shown promising clinical results are nucleoside analogs. However, the success of the nucleoside analogs is limited by their toxicity and the ability of the HIV virus to develop drug resistant mutations in reverse transcriptase. The current proposal seeks to continue our mechanistic studies of RT to more complex physiologically relevant substrates and systems including natural tRNA3Lys and NCp7 protein. These studies will continue to focus on increasing our mechanistic understanding, at a molecular level, of the enzymatic activities of RT and the interaction with clinically significant nucleoside inhibitors, the mechanism of resistance to these compounds that develops through specific RT mutations, the mechanism of differential selectivity between the inhibition of HIV RT and the mitochondrial DNA polymerase as it relates to cytotoxicity, and finally to explore the molecular basis of synergy between nucleoside (N-RTIs) and nonnucleoside reverse transcriptase inhibitors (NNRTIs). Continuation of these aims will contribute to our overall understanding and development of a SAR (structure activity relationship) that correlates structural and stereochemical features of nucleoside analog drugs with their kinetic and mechanistic behavior toward relevant HIV-1 RT and polymerase targets. It is the ultimate goal of these studies to develop an in-depth understanding of the enzyme mechanism of HIV 1-RT and the interaction with inhibitors as well as to uncover the underlying mechanisms of drug resistance and toxicity that could ultimately lead to compounds that are less toxic, more selective, and hence more effective as therapeutics.